PROJECT SUMMARY/ABSTRACT We discovered a novel family of atypical secretory pathway kinases that phosphorylate secreted proteins. One of these kinases, Fam20C, targets SxE/pS motifs on secreted proteins. Approximately 75% of human serum, plasma and cerebrospinal fluid phosphoproteins are phosphorylated on the Fam20C consensus motif, underscoring the importance of this discovery. We used comparative phosphoproteomics to identify over 100 secreted substrates of Fam20C, demonstrating that Fam20C generates the majority of the secreted phosphoproteome. While Fam20C plays a critical role in tissue mineralization, the presence of Fam20C in invertebrates that lack mineralized tissue, as well as the diverse functions of Fam20C substrates, suggest that this kinase has additional roles. Notably, one ancestral substrate of Fam20C is egg yolk vitegllogenin, a secreted phosphoprotein of the Large Lipid Transport Protein (LLTP) family. While vitellogenins are unique to egg-laying animals, LLTP proteins in humans have central roles in lipid transport and include the apolipoproteins and the microsomal triglyceride transfer protein. We found that five human apolipoproteins, APOA2, APOB, APOE, APOL1 and APOA5, as well as proprotein convertase subtilisin type 9 (PCSK9) are all substrates of Fam20C in humans. These results lead us to hypothesize that regulation of lipid trafficking may be one of the major, and possibly the ancestral function of Fam20C. We will test this hypothesis through in vitro and in vivo experiments to establish direct kinase-substrate relationships between Fam20C and PCSK9 or lipoproteins. We will use cell- based assays to specifically study how phosphorylation affects the secretion, processing, and biological activity of these substrates. We have developed a liver-specific Fam20C knockout mouse and will undertake an unbiased mass spectrometry-based lipidomics approach to determine the in vivo contribution of Fam20C to lipid homeostasis. The biological mechanisms responsible for distribution of lipids throughout the body are of fundamental importance to human health and disease, including the massive public health burden of atherosclerosis and heart disease.